Performing a Separation with Sepharose® High Performance: Purification with High Resolution

Correct sample and buffer preparation is essential in order to achieve optimal separation and avoid any deterioration in column performance. Samples must be fully dissolved and free from particles or other material likely to interfere with the separation. Refer to Appendix 1 for recommendations and advice on sample preparation.

Filter buffers and samples after all salts and additives have been included. Use high-quality water and chemicals. Filter solutions using 0.45 μm or 0.22 μm filters. To avoid formation of air bubbles in a packed column and to ensure reproducible results, the column and buffers should be at the same temperature when preparing for a run.

To avoid problems with precipitation, check the salt stability window of the sample components. Avoid working at concentrations near to the stability limit of the target protein. For samples with unknown hydrophobic properties, try the following:

start buffer: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 elution buffer: 50 mM phosphate buffer, pH 7.0

Note that flow rates may need to be reduced due to the viscosity of the chosen start buffer, sample characteristics, loading requirements and the equipment used.

First-time use or after long-term storage

1. To remove ethanol, wash with 5 column volumes of distilled water or elution buffer.

Flow: 1 ml/min, HiTrap™ 1 ml

5 ml/min, HiTrap™ 5 ml

0.8 ml/min, HiLoad™ 16/10, 20 ml

2.2 ml/min, HiLoad™ 26/10, 53 ml

25 cm/h for Sepharose® High Performance packed in larger columns

2. Wash with 5 column volumes of start buffer.

Flow: 1 ml/min, HiTrap™ 1 ml

5 ml/min, HiTrap™ 5 ml

3 ml/min, HiLoad™ 16/10, 20 ml

8 ml/min, HiLoad™ 26/10, 53 ml

50 cm/h for Sepharose® High Performance packed in larger columns

3. Perform a blank elution to check conductivity.

Separation by gradient elution

Flow: 1 ml/min, HiTrap™ 1 ml

5 ml/min, HiTrap™ 5 ml

3 ml/min, HiLoad™ 16/10, 20 ml

8 ml/min, HiLoad™ 26/10, 53 ml

50–100 cm/h for Sepharose® High Performance packed in larger columns

Collect fractions throughout the separation.

  1. Equilibrate column with 5–10 column volumes of start buffer or until the UV baseline and conductivity are stable.
  2. Adjust the sample to the chosen salt concentration (and pH if necessary). Filter and apply to the column.
  3. Wash with 5–10 column volumes of start buffer or until the UV baseline and conductivity are stable so that all unbound material has washed through the column.
  4. Begin elution using a gradient volume of 10–20 column volumes, increasing the proportion of elution buffer until the salt concentration reaches a minimum, that is, salt-free buffer.
  5. Wash with 2–5 column volumes of salt-free elution buffer to elute remaining hydrophobically bound material.
  6. Re-equilibrate with 5–10 column volumes of start buffer or until conductivity reaches the required value.

Separation by step elution

Flow: 1 ml/min, HiTrap™ 1 ml

5 ml/min, HiTrap™ 5 ml

3 ml/min, HiLoad™ 16/10, 20 ml

8 ml/min, HiLoad™ 26/10, 53 ml

50100 cm/h for Sepharose® High Performance packed in larger columns

Collect fractions throughout the separation.

  1. Equilibrate the column with 5–10 column volumes of start buffer or until the baseline, eluent pH and conductivity are stable.
  2. Adjust the sample to the chosen salt concentration (and pH if necessary). Filter and apply to the column.
  3. Wash with 5–10 column volumes of start buffer or until the UV baseline and conductivity are stable so that all unbound material has washed through the column.
  4. Elute with 5 column volumes of elution buffer + salt at chosen concentration.
  5. Repeat step 4 at lower salt concentrations until the target protein(s) has been eluted.
  6. Wash with 2–5 column volumes of salt-free elution buffer to elute remaining hydrophobically bound material.
  7. Re-equilibrate with 5–10 column volumes of start buffer or until conductivity reaches the required value.

Save time by using higher flow rates during the salt-free wash and re-equilibration steps, but do not exceed the maximum recommended flow for the medium.

Check column performance regularly by determining column efficiency and peak symmetry. See Appendix 2.

Never leave columns or equipment in high salt solutions.

Cleaning

Correct preparation of samples and buffers and application of a salt-free buffer at the end of each separation should keep most columns in good condition. However, reduced performance, a slow flow rate, increasing back pressure or complete blockage are all indications that the medium needs to be cleaned using more stringent procedures in order to remove contaminants.

Whenever possible, reverse the direction of flow during cleaning so that contaminants do not pass through the entire column length. The number of column volumes and time required for each cleaning step may vary according to the degree of contamination. If the cleaning procedure to remove common contaminants does not restore column performance, change the top filter (when possible) before trying alternative cleaning methods. Care should be taken when changing a filter as this may affect the column packing and interfere with performance.

The following procedure should be satisfactory to remove common contaminants such as precipitated proteins.

Flow: 1 ml/min, HiTrap™ 1 ml

5 ml/min, HiTrap™ 5 ml

3 ml/min, HiLoad™ 16/10, 20 ml

8 ml/min, HiLoad™ 26/10, 53 ml

40 cm/h, with a contact time of 1–2 hours, for Sepharose® High Performance packed in larger columns

Note that flow rates may need to be reduced due to the condition of the column and the viscosity of the sample, buffers or storage solutions.

  1. Wash with up to 4 column volumes of 1 M NaOH.
  2. Wash with at least 3 column volumes of water or until eluent pH is neutral.
  3. To start a new separation: re-equilibrate with at least 3 column volumes of start buffer or until the correct eluent pH is achieved.
  4. For storage: wash with at least 5 column volumes of storage solution. Allow UV baseline to stabilize before storing the column.

To remove lipids, lipoproteins and very hydrophobic proteins, see Appendix 1.

Media characteristics

Composition: Sepharose® High Performance is based on highly cross-linked, 6% agarose formed into spherical particles (mean size 34 μm) and substituted with hydrophobic ligands via uncharged, chemically stable O-ether linkages.

Table 11. Characteristics of Phenyl and Butyl Sepharose® High Performance.

Product Matrix Ligand density per ml medium pH stability* Mean particle size
Phenyl Sepharose® High Performance 6% cross-linked agarose, spherical particles 25 μmol Long term: 3–13

 

Short term: 2–14

34 μm
Butyl Sepharose® High Performance† 6% cross-linked agarose, spherical particles not determined Long term: 3–13

 

Short term: 2–14

34 μm

*Long-term pH stability refers to the pH interval where the medium is stable over a long period of time without adverse side effects on the chromatography performance.

Short-term pH stability refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

All ranges are estimates based on the experience and knowledge within Cytiva.

† Available as a Custom Designed Medium.

Chemical stability

For daily use, Sepharose® High Performance is stable in all common, aqueous buffers, 1 M NaOH, denaturing agents (8 M urea, 6 M guanidine hydrochloride), 70% ethanol, 1 M acetic acid, 30% isopropanol, 30% acetonitrile and up to 2% SDS.

Storage

For column storage, wash with 5 column volumes of distilled water followed by 5 column volumes of 20% ethanol. Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid overpressuring the column. Ensure that the column is sealed well to avoid drying out. Whenever possible, use a storage and shipping device, if supplied by the manufacturer. Store columns and unused media at 4°C to 30°C in 20% ethanol. Do not freeze.

Large-scale production columns are often stored in 0.01 M sodium hydroxide as an alternative to 20% ethanol.

To avoid the formation of air bubbles, ensure that columns, buffers and equipment are at the same temperature before use.

Materials